Viral and bacterial diseases in fin-fish, shellfish or other aquatic lifeforms pose a serious problem for the aquaculture industry. Owing to the high density of animals in the hatchery tanks or enclosed marine farming areas, infectious diseases may eradicate a large proportion of the stock in, for example, a fin-fish, shellfish, or other aquatic lifeforms facility. Prevention of disease is a more desired remedy to these threats to fish than intervention once the disease is in progress. Vaccination of fish is the only preventative method which may offer long-term protection through immunity.
The fish immune system has many features similar to the mammalian immune system, such as the presence of B cells, T cells, lymphokines, complement, and immunoglobulins. Fish have lymphocyte subclasses with roles that appear similar in many respects to those of the B and T cells of mammals. Additionally, the efficiency of the immune response of fish can be affected by outside stresses, as is true in mammals. However, fish, unlike mammals, display a temperature-dependent development of protective immunity in response to antigens.
Most vaccines for fish have been developed against bacteria while there have been very few fish vaccines made for combating viral or parasitic diseases. Fish have been immunized by antigen-based immunization methods using live attenuated pathogens, killed whole pathogens, or more recently, in laboratory settings, recombinant proteins. While live attenuated vaccines induce good humoral and cell-mediated immune responses and can be administered orally or by immersion or injection, there is the important risk of reversion to a virulent form. Whole live attenuated vaccines are not preferred in industrial farming due to the risk of contaminating other fish--a live attenuated vaccine which may be generally safe for the target species of fish may be virulent in other species of fish.
Fish vaccines using whole killed bacteria (i.e. bacterins) or recombinant proteins from pathogens expressed in cell lines (subunit vaccines) have the disadvantage of inducing short-lived immune responses. Injected antigen, including recombinant protein, is processed solely in an exogenous form usually causing induction of a humoral response (i.e., production of antibodies) but often a failure to induce cell-mediated immunity (i.e., cytotoxic T-cells).
Another disadvantage of whole killed and subunit vaccines is that they almost always must be injected and they require an adjuvant to induce an effective immune response. Intramuscular injections of these adjuvants can cause granuloma formation which scars the flesh and lowers the market value of the fish. Intraperitoneal injection of adjuvants may cause adhesions between the viscera which can affect the health of the fish and retard fish growth.
Recombinant protein vaccines are difficult and expensive to make especially if the protein must be purified. For example, bacterially-expressed recombinant proteins may form inclusion bodies from which recovery of protein in correct configuration may be low or nonexistent. Induction of an immune response may require that the antigenic protein be correctly glycosylated and folded, which may not be accomplished in a cell other than an animal cell.
Some of the current methodologies for administering vaccines are not technically or economically practical. For example, direct injection of recombinant and whole killed pathogen vaccines into the fish is labor intensive and expensive relative to the future market value of the fish. Furthermore, injection needles can cross-infect fish with contaminating pathogenic organisms, and accidental injection of humans can cause severe or fatal infections and anaphylactic reactions. Moreover, noninjurious injection of small fish is very difficult, especially in young fry, which are particularly susceptible to disease.
A less expensive and easier method which has been used to administer killed viral or bacterial vaccines is an oral method wherein the vaccine is added directly to the water or incorporated into fish food. Oral vaccines have historically shown inconsistent and relatively low levels of protection suggesting that they may be best used as a method of revaccination.
Genes have been introduced directly into animals by using live viral vectors containing particular sequences from an adenovirus, an adeno-associated virus, or a retrovirus genome. The viral sequences allow the appropriate processing and packaging of a gene into a virion, which can be introduced to animals through invasive or non-invasive infection. Viral vectors have several disadvantages. Viral vectors being live pathogens, still carry the risk of inadvertent infection. Furthermore, proteins from viral vector sequences induce undesirable inflammatory or other immune responses which may prevent the possibility of using the same vector for a subsequent vaccine or boost. Viral vectors also limit the size of the target gene that can be expressed due to viral packaging constraints.
Naked DNA transfects relatively efficiently if injected into skeletal muscle but poorly or not at all if injected into other tissues (Wolff et al., Science 247:1465-1468 (1990), incorporated herein by reference). Plasmid DNA coated onto the surface of small gold particles and introduced into the skin by a helium-driven particle accelerator or "gene-gun" can directly transfect cells of the epidermis and dermis (Pecorino and Lo, Current Biol., 2:30-32 (1992), which is incorporated herein by reference).
DNA has also been introduced into animal cells by liposome-mediated gene transfer. DNA-liposome complexes, usually containing a mixture of cationic and neutral lipids, are injected into various tissues or instilled into the respiratory passages. Nabel et al., Hum. Gene Ther., 3:649-656 (1992), which is incorporated herein by reference, have shown that liposomes may be used to transfect a wide variety of cell types by intravenous injection in mammals. In addition, liposome-mediated gene transfer has been used to transfer the cystic fibrosis transmembrane conductance gene into the nasal epithelium of mice and humans suffering from cystic fibrosis (Yoshimura et al., Nucleic Acids Reg., 12:3233-3240 (1992) and Caplan et al., Nature Med., 1:39-46 (1995), respectively, both of which are incorporated herein by reference.
Substances may also be administered using biodegradable microspheres composed of polymers such as polyester poly(lactide-co-glycolide) (Marx et al., Science, 260:1323-1328 (1993), incorporated herein by reference). It is notable that these particles can survive the upper digestive system and arrive intact in cells of gut-associated lymphoid tissue (Eldridge et al., Adv. Exp. Med. Biol., 251:191-202 (1989), incorporated herein by reference). Biodegradable microspheres have been used to deliver recombinant antigens, toxoids or attenuated virus into mammals by systemic and oral routes (O'Hagan et al., Immunology 73:239-242 (1991); O'Hagen et al., Vaccine 11:149-154 (1993); Eldridge et al., Mol. Immunol. 228:287-293 (1991) incorporated herein by reference). They may also be useful to deliver recombinant plasmid DNA to gut-associated lymphoid tissue for the purpose of immunization.
While most work has been carried out on mammals, plasmid DNA encoding reporter genes have been successfully introduced into fish by intramuscular injection (Hansen et al., FEBS Lett. 290:73-76 (1991), incorporated herein by reference). Thus, cells in fish can express proteins from a foreign gene with the same types of vector constructs (i.e., backbones, promoter and enhancer elements) that are used in mammals.
The induction of an immune response to a protein expressed from an introduced gene was first suggested by Acsadi et al., New Biologist 3:71-81 (1991), which is incorporated herein by reference, who found that after plasmid DNA transfer into rat cardiac muscle, reporter gene expression was transient but could be prolonged by treatment with an immuno-suppressant. Subsequently, it was shown that antibodies were induced in rodents against human growth hormone (Tang et al., Nature, 356:152-154 (1992); Eisenbraun et al., DNA Cell. Biol., 12:791-797 (1993), both of which are incorporated herein by reference) or human .alpha.-antitrypsin (Tang et al., Nature, 356:152-154 (1992), also incorporated herein by reference) when these proteins were expressed from DNA coated onto gold particles and introduced into cells of the skin by bombardment.
DNA-based immunization refers to the induction of an immune response to an antigen expressed in vivo from a gene introduced into the animal. This method offers two major advantages over classical vaccination in which some form of the antigen itself is administered. First, the synthesis of antigen in a self-cell mimics in certain respects an infection and thus induces a complete immune response but carries absolutely no risk of infection. Second, foreign gene expression may continue for a sufficient length of time to induce strong and sustained immune responses without boost.
Several mammalian animal models of DNA-based immunization against specific viral, bacterial or parasitic diseases have been reported. These include influenza [(Fynan et al., Proc. Nat'l Acad. Sci. USA, 30 90:11478-11482 (1993); Montgomery et al., DNA Cell. Biol., 12:777-783 (1993); Robinson et al., Vaccine, 11:957-960(1993); Ulmer et al., Science, 259:1745-1749 (1993)], HIV [Wang et al. (1993)], hepatitis B [Davis et al., Hum. Molec. Genet., 2:1847-1851 (1993)], malaria [Sedagah et al., Proc. Nat'l Acad. Sci., USA, 91:9866-9870 (1994)], bovine herpes [(Cox et al., J. Virol, 67:5664-5667 (1993)], herpes simplex [Rousse et al., J. Virol., 68:5685-5689 (1994); Manicken et al. J. Immunol., 155:259-265 (1995)], rabies [Xiang et al., Viroloay, 199:132-140 (1994)]; lymphocytic choriomeningitis [Yokoyama et al., J. Virol., 6964:2684-2688 (1995)] and tuberculosis [Lowrie et al., Vaccine, 12:1537-1540 (1994)], all of which are incorporated herein by reference. In most of these studies a full-range of immune responses including antibodies, cytotoxic T lymphocytes (CTL), T-cell help and (where evaluation was possible) protection against challenge was obtained. In these studies naked DNA was introduced by intramuscular or intradermal injection with a needle and syringe or by instillation in the nasal passages, or the naked DNA was coated onto gold particles which were introduced by a particle accelerator into the skin.
There is a need for novel systems to vaccinate fin-fish, shellfish, and other aquatic animals against diseases. These systems should be inexpensive to produce and administer, avoid the use of live, attenuated organisms, and induce strong and long-lasting immunity preferably without boost and with induction of both antibodies and cell-mediated immunity. More preferably, the system should be applicable to small fish, be less stressful to fish during administration, and have the capacity of simultaneously immunizing many animals for reduced labor-related costs.